Origins and characteristics of dissolved organic matter fueling harmful dinoflagellate blooms revealed by δ13C and d/l-Amino acid compositions

We measured the concentrations of dissolved inorganic and organic nutrients, dissolved organic carbon (DOC), total hydrolyzable amino acids (THAA), fluorescent dissolved organic matter (FDOM), phytoplankton pigments, and δ13C-DOC during the summer of 2019 in the harmful dinoflagellate bloom regions of the southern coast of Korea. In the harmful dinoflagellate bloom region, the concentrations of inorganic nitrogen were depleted, inhibiting the growth of diatoms, while the concentrations of dissolved organic components (nutrients, DOC, FDOM, and amino acids) which fuel dinoflagellates were unusually high. Thus, we attempted to investigate the origins and characteristics of DOM which fuels the harmful dinoflagellate blooms. The δ13C-DOC values (− 22.2‰ to − 18.2‰) indicate that the elevated DOC concentrations result from in-situ biological production rather than terrestrial inputs. The enantiomeric (D/L) ratios of THAA indicate that dissolved organic nitrogen was more labile in the early stage of harmful dinoflagellate bloom and became more refractory in the final stage. Our results suggest that the marine production of bioavailable DOM plays an important role in initiating and sustaining harmful dinoflagellate blooms.

The outbreak of harmful algal blooms in the coastal ocean associated with increased inputs of nutrients has been reported worldwide 1,2 . In particular, dinoflagellate blooms often have serious effects on aquaculture and wild organisms as they cause massive kills of fish and other invertebrate by producing toxins or mucus substances 3,4 . The environmental conditions in harmful dinoflagellate bloom (HDB) regions have been characterized by low concentrations of dissolved inorganic nutrients and high concentrations of dissolved organic nutrients [5][6][7][8] . Many culture experiments and field observations showed that this condition is favorable for the growth of dinoflagellates in competition with diatoms since HDB forming species (e.g., Margalefidinium, Alexandrium) are capable of converting organic nitrogen compounds to inorganic nutrients, while the growth of diatoms is limited under low inorganic nutrient concentrations [8][9][10][11][12][13] .
Many studies were conducted to determine the characteristics of dissolved organic matter (DOM) in HDB regions. The concentrations of dissolved organic carbon (DOC) in the bloom regions were significantly higher than those of non-bloom regions in Yeosu and Tongyeong of Korea 14,15 . The concentrations of fluorescent dissolved organic matter (FDOM) which is commonly composed of marine humic-like (FDOM M ), terrestrial humiclike (FDOM C ), and protein-like (FDOM T ) components also increased in HDB areas 14,16,17 . FDOM, which contributes about 20-70% of the DOC in coastal waters 18 , is known to play an important role in the outbreak of HDBs as it protects organisms from UV radiation and becomes an energy source for the growth of dinoflagellates [18][19][20] .
In HDB regions, DOM originates from various sources including rivers and groundwater as well as in-situ production 15,21 . In general, humic-like FDOM originate mainly from terrestrial sources 18,22 , although Kwon et al. 14 showed in-situ production of FDOM C in HDB regions. FDOM T , which is mostly labile, is produced freshly by www.nature.com/scientificreports/ from the local sources 5,8 . In addition, the Seomjin River (drainage area: 4896 km 2 ) influences the salinity and the nutrient concentrations in this region, mainly during the summer monsoon season.
In the southern coast of Korea, Margalefidinium polykrikoides (formerly Cochlodinium polykrikoides) is the dominant species causing HDBs (hereafter red tides) 35 . In this region, red tides have occurred repeatedly since 1982. In the southern sea of Korea, the inputs of dissolved inorganic nitrogen (DIN) from heavy rainfall events 36 and the Yangtze River diluted water 37 had been attributed to the main sources of nutrients. However, Lee and Kim 5 showed that local sources, such as submarine groundwater discharge, were the main source of nutrients fueling red tides. The rapid growth of M. polykrikoides in competition with diatoms happened under the condition of depleted DIN or dissolved inorganic phosphorus (DIP) and enriched organic nutrients, when the supply of inorganic nutrients was halted 5,6,8,38,39 . In general, M. polykrikoides patches formed in offshore waters are transported to nearshore waters and gradually accumulated as the abundance of competing phytoplankton species (i.e., fast-growing diatoms) is low 13 .
In the summer of 2019, M. polykrikoides red tides were first observed in the study region on August 23 and then spread to the wide area of the study region (http:// www. nifs. go. kr/) 40 . The red tide disappeared around September 24. The seawater temperatures of the study area ranged from 23 to 26 °C during the red tide outbreak periods, which were within the optimum temperature range (21-26 °C) for the growth of M. polykrikoides 41 . The salinity ranged from 30 to 34 psu throughout the study period. The patches of red tide were mostly located in proximity to the shore, perhaps associated with passive accumulation by geographical and tidal features in this region (Fig. 1). Seawater samples were collected in the surface layer (~ 0.5 m depth) using a submersible pump on shipboard from 2017 to 2019. Samples for DOC and FDOM were collected for all the sampling periods. Additionally, in 2019, samples for dissolved inorganic/organic nutrients were collected from May to September. Samples for δ 13 C-DOC and amino acids were collected over three periods in 2019 (before the outbreak: August 14-16, the early stage: August 23-27, and the final stage: September 17). Samples for the analysis of phytoplankton pigments were collected during the final stage of red tide (September 17, 2019). Salinity and temperature were measured in-situ using a portable multimeter (Orion Star A329, Thermo Scientific, USA). All seawater samples for the dissolved form were filtered through pre-combusted (450 °C, 5 h) glass-fiber filters (Whatman GF/F, pore size: 0.7 µm). The filter samples were used to measure the concentrations of phytoplankton pigments. Sample analyses were completed mostly within a week from the sample collection.

Sampling.
Analyses. Phytoplankton pigment analyses. The filter samples for pigment analysis were stored at − 80 °C.
The filter samples were extracted in 95% methanol, with an internal standard (canthaxanthin), at 4 °C for 24 h in the dark. Extracts were sonicated (5 min) and centrifuged (3500 rpm, 10 min) and then filtered through PTEE membrane filters (pore size: 0.2 µm) to remove residual particles. The extracts were analysed for pigments by using high-performance liquid chromatography (HPLC) (Waters 2695) with a Waters Symmetry C8 column (4.6 × 150 mm, particle size: 3.5 μm, pore size: 100 Å) according to the method described by Zapata et al. 42 . Identification and quantification of chlorophyll a (chl. a), fucoxanthin (marker pigment for diatom), and peridinin (marker pigment for dinoflagellate) were based on their retention times with authentic standards (DHI Inc., Denmark). were measured by an auto nutrient analyzer (New QuAAtro39, Seal Analytical, Germany). The analytical accuracy was verified with reference seawater materials (KANSO Technos, Japan). Dissolved total phosphorus (DTP) and DTN were analysed using the same instrument after chemical oxidation by potassium persulfate at 120 °C for 30 min 43 . The concentrations of dissolved organic phosphorus (DOP) and DON were calculated by subtracting the measured DIP and DIN from the measured DTP and DTN concentrations, respectively.
FDOM analyses. The seawater samples for FDOM were stored at 4 °C in pre-combusted (450 °C, 5 h) amber vials. The FDOM was measured by a spectrofluorometer (Aqualog, Horiba, USA). The scanning wavelength for fluorescence excitation-emission matrices (EEMs) was 252-600 nm in 2 nm increments for excitation (Ex) and 211-616 nm in 3 nm increments for emission (Em) with an integration time of 3 s. Raman and Rayleigh scattering signals, the inner-filter effect, and blank subtraction were corrected using the SOLO software (Eigenvector Research Inc., WA, USA). The fluorescence intensity was normalized by the Raman peak area of ultrapure water and referred to as Raman units (R.U.) 44 . The FDOM analyses with the parallel factor analysis (PARAFAC) model identified one protein-like fluorescence peak and two humic-like fluorescence peaks, as described by Coble 18 . Component 1 (Ex/Em = 278/340 nm), component 2 (Ex/Em = 362/466 nm), and component 3 (Ex/ Em = 338/400 nm) were found to be FDOM T (tryptophan-like), FDOM C , and FDOM M , respectively. The PARA-FAC model results were validated by random initialization and split-half analysis 45  www.nature.com/scientificreports/ DOC and δ 13 C-DOC analyses. The seawater samples for DOC and δ 13 C-DOC were stored at room temperature in pre-combusted glass ampoules (450 °C, 5 h) after being acidified with 6 M HCl. The DOC concentration was determined by a high-temperature catalytic oxidation (HTCO) method using a total organic carbon (TOC) analyzer (TOC-L, Shimadzu, Japan) 46,47 . Verification of analytical accuracy was performed using a reference material (~ 43 μM; University of Miami, USA). The δ 13 C-DOC values were measured by an isotope ratio mass spectrometer (IRMS; Isoprime, Elementar, Germany) connected to a TOC analyzer (Vario TOC cube, Elementar, Germany) via an interface system (isoTOC interface, Elementar, UK) 48 . The δ 13 C-DOC values were verified with the reported values of the reference materials: IAEA-CH6 sucrose (δ 13 C = − 10.45 ± 0.03‰), Suwannee River fulvic acid (δ 13 C = − 27.6 ± 0.12‰; International Humic Substances Society), and deep-seawater reference (University of Miami, USA) as previously stated in Lang et al. 49 (δ 13 C = − 21.7 ± 0.3‰) and Panetta et al. 50 (δ 13 C = − 21.4 ± 0.3‰).
Amino acid analyses. The seawater samples for amino acids were stored at − 20 °C. The d-and l-enantiomers of amino acids were analysed as described by Dittmar et al. 51 . The seawater samples were hydrolyzed with 12 M HCl and 11 mM ascorbic acid at 110 °C for 24 h after flushing with ultra-pure nitrogen. After hydrolysis, amino-acid enantiomers were derivatized with o-phthaldialdehyde and N-isobutyryl-l-cysteine. The derivatized samples were measured by a HPLC system equipped with an Alltima HP C18 column (particle size: 5 µm, 4.6 × 150 mm) and a Waters 2475 fluorescence detector (Ex/Em: 330/445 nm). A total of 13 individual amino acids were included in the analysis: serine (Ser), glutamic acid (Glu), aspartic acid (Asp), alanine (Ala), threonine, glycine, arginine, tyrosine, valine, phenylalanine, leucine, isoleucine, and γ-amino butyric acid (GABA). During the hydrolysis, glutamine and asparagine are converted to glutamic acid and aspartic acid, respectively. Therefore, Glu refers to the sum of glutamic acid and glutamine, and Asp refers to the sum of aspartic acid and asparagine. The concentrations of d-enantiomers and l-enantiomers of Ser, Glu, Asp, and Ala were averaged, respectively, for the d-and l-amino acid values in this study.

Results
During  (Fig. 2). The average concentrations of chl. a and peridinin in the patch areas were 2.8-and 4.5-fold higher than those in the non-patch areas, respectively, while the concentrations of fucoxanthin in the patch areas were 0.7-fold lower than those in the non-patch areas ( Supplementary Fig. S5). During all sampling periods in 2019 (before and after the red tide outbreak), the concentrations of DIN and DIP ranged from 0.0 to 3.3 µM and from 0.02 to 0.53 µM, respectively (Fig. 3). In general, the concentrations of DON (3.0-13.0 µM) and DOP (0.07-0.85 µM) were much higher than those of DIN and DIP for the same samples (Fig. 3). Overall, the concentrations of DIN during the red tide periods were significantly lower than those before the red-tide outbreak periods (Kruskal-Wallis: p = 0.001) (Fig. 3). Although the average concentrations of DON in the patch areas (8.9 ± 2.3 µM) were slightly higher than those in the non-patch areas (7.6 ± 2.4 µM), it did not show a significant statistical difference (t test: p = 0.381) (Fig. 3). On the other hand, the concentrations of DOP showed no significant spatial and temporal changes (Kruskal-Wallis: p = 0.494) (Fig. 3). In general, the spatial distributions of higher DON and lower DIN concentrations along the coast during the red tide period of September 17 coincided with the higher peridinin concentration areas (Supplementary Fig. S5).
The concentrations of DOC during all the sampling periods in 2019 ranged from 71 to 137 µM (average: 90 ± 13 µM) (Fig. 3). In general, higher concentrations of DOC were observed in the low salinity sites before the outbreak of red tides (Supplementary Figs. S1, S2, and S3). However, during the red tide periods, the concentrations of DOC were significantly higher than those before the outbreak of red tides (Kruskal-Wallis: p < 0.001) (Fig. 3). The concentrations of DOC in the patch areas (average: 113 ± 14 µM) were 1.2-fold higher than those in the non-patch areas (average: 96 ± 9 µM) during the red tide periods (t test: p = 0.001). The values of δ 13 C-DOC ranged from − 22.2 to − 18.2‰ for all sampling sites over three periods (before the outbreak: August 14-16, the early stage: August 23-27, and the final stage: September 17) (Fig. 4).
The  (Fig. 3). Similar to the spatial distributions of DOC, the concentrations of FDOM C were generally higher in the low salinity sites before the red tide outbreak (Supplementary Figs. S1, S2, and S3). The concentrations of FDOM C during the red tide periods were significantly higher than those before the outbreak periods (Kruskal-Wallis: p < 0.001) (Fig. 3), while FDOM T showed no clear temporal trend (Fig. 3). www.nature.com/scientificreports/ The concentrations of FDOM M were higher during the red tide periods than those before the red tide outbreak (Kruskal-Wallis: p < 0.001) (Fig. 3). During the red tide periods, the average concentrations of FDOM C and FDOM T in the patch areas (average: 1.76 ± 0.04 R.U., 1.96 ± 0.04 R.U., respectively) were 1.7-and 1.3-fold higher than those in the non-patch areas (average: 1.01 ± 0.43 R.U., 1.52 ± 0.23 R.U., respectively) (Mann-Whitney U test: p < 0.001, p = 0.008, respectively), respectively (Fig. 3). The concentrations of THAA, l-AA, and d-AA were in the range of 169-1028 nM, 82-495 nM, and 4-31 nM, respectively, for all sampling sites over the three periods in 2019 (August 14-16, August 23-27, and September 17) (Fig. 5). The average concentrations of these components in the early stage of red tide (average: 536 ± 168 nM for THAA, 268 ± 77 nM for l-AA, and 19 ± 8 nM for d-AA) were about a factor of two higher than those during the other stages (Fig. 5). However, there was no significant difference in the average concentrations of THAA, l-AA, and d-AA between the before-outbreak period and the final stage period (Fig. 5). The averaged D/L ratios of amino acids ranged from 0.02 to 0.15, which increased gradually according to the red tide stages (one-way ANOVA: p = 0.008) (Fig. 5). Except for Ala (Kruskal-Wallis: p = 0.647), D/L ratios of individual amino acids (Asp, Glu, and Ser) also increased gradually according to the red tide stages (one-way ANOVA: p < 0.001, one-way ANOVA: p = 0.007, Kruskal-Wallis: p = 0.005, respectively) ( Supplementary Table S2). Moreover, there was no significant difference in the D/L ratios of both averaged and individual amino acids between the patch and nonpatch areas during the red tide periods (t-test for averaged, Asp, and Glu and Mann-Whitney U test for Ala and Ser: p > 0.05) (Supplementary Table S2). The ratios of Glu/GABA ranged from 3 to 20 for all sampling sites over the three periods (August 14-16, August 23-27, and September 17). The ratios of Glu/GABA were higher in the early stage of red tide (average: 13 ± 4) than those for the other periods (one-way ANOVA: p = 0.021) (Fig. 5). However, there was no significant difference in the ratios of Glu/GABA between the patch and non-patch areas during the red tide periods (t test: p = 0.921) (Supplementary Table S2).

Discussion
General characteristics of red tide areas. Peridinin and fucoxanthin are diagnostic indices for dinoflagellates and diatoms, respectively 53 . The concentrations of chl. a and peridinin during the red tide period of 2019 were 3.6-fold and 2.1-fold lower than those during the massive red tide outbreak of 2014, respectively 8 (Fig. 2). However, the concentration of peridinin in 2019 was 6-fold higher than that in 2017 when no red tide outbreaks were observed 8 (Fig. 2). The concentrations of fucoxanthin showed the highest value during the nonoutbreak period 8 (Fig. 2). This difference in peridinin concentrations seems to be associated with the magnitude of red tides in different years: 2014 (~ 1.5 × 10 4 cells mL −1 ), 2017 (non-outbreak), and 2019 (~ 5.8 × 10 3 cells mL −1 ) (http:// www. nifs. go. kr/) 40 .
During the red tide periods, except for one station, the concentrations of DIN were depleted (< 2 µM), and DIN/DIP ratios were lower than 11 (Fig. 3), indicating the limitation of biological production by DIN. However, higher DON concentrations were observed during the red tide periods (Fig. 3), consistent with those in previous

Origins of DOC and FDOM in red tide areas.
In river-dominated coastal oceans, the distribution of DOC is generally dependent on salinity 54,55 . However, in this study region, the distribution of DOC concentrations was independent of the salinity, with enhanced DOC concentrations in the patch areas (Fig. 3). This trend is consistent with the previous observations in this region from different years (i.e., from 2013 to 2018) (Fig. 6). A previous culture experiment showed that M. polykrikoides significantly release various organic substances, such as polysaccharides, proteins, amino acids, and carbohydrates 56 . Thus, the concentrations of DOC were higher in the patch areas than in the non-patch areas (Fig. 3). The values of δ 13 C-DOC (ranged from − 22.2 to − 18.2‰) fall into the range of marine sources of DOC (ranged from − 22 to − 18‰), indicating that the main source of DOC in the red tide areas is a marine origin (Fig. 4). This result is consistent with the previous hypothesis 8 that DOM fueling red tides in this region is produced mainly by microbial production following the massive diatom growth 57,58 and abrupt cessation of DIN supply. www.nature.com/scientificreports/ In general, the major source of FDOM C in coastal waters is known to originate mainly from terrestrial freshwater, as shown by significant negative correlations between FDOM C and salinity 59,60 . As such, in Gwangyang Bay, which is located between Yeosu and Namhae, Korea, FDOM C concentrations showed significant negative correlations (r 2 = 0.92, p < 0.0001) against salinity 61 . However, the distribution of FDOM C in this study region was independent of the salinity, similar to that of DOC (Fig. 3). Our observations show that the concentrations of both FDOM C and FDOM T were higher in the patch areas (Fig. 3). Culture experiments have shown that FDOM C can be produced directly from phytoplankton and microbial transformation of planktonic materials 62 . Also, FDOM T is known to be produced by biological activities 63,64 . Previous field observations in the same region also showed the relatively enhanced concentrations of FDOM C and FDOM T during the red tide periods compared with those for the non-outbreak periods (Fig. 6). Thus, it is likely that FDOM C and FDOM T are produced in the red tide region as suggested by Kwon et al. 14 .
In contrast, there was no significant difference in the concentrations of FDOM M between the patch and nonpatch areas (Fig. 3), though the distribution pattern of FDOM M was generally similar to that of FDOM C 22,48,65 . FDOM M can be produced either by bacterial activities 65 or phytoplankton 63 and also can be taken up by bacteria 66 . In general, the consumption rates of FDOM M by bacteria are approximately 2-fold higher than the production rates by phytoplankton 66 . Thus, the preferential consumption of FDOM M by bacteria in red tide areas where higher production occurs could result in similar concentrations in both red tide periods and non-red tide periods (Fig. 6), as previously shown by Kwon et al. 14 .

Characteristics of DOM revealed by amino-acid indices.
Most of the amino acids have enantiomeric forms with l-and d-enantiomers in seawater 67 . While the proteins are composed of l-enantiomers in most organisms, d-enantiomers are known to be mostly derived from bacterial cell membranes. In general, d-enantiomers tend to be accumulated during DOM degradation because of the key role of bacteria in DOM degradation 68,69 . A higher enantiomeric (D/L) ratio of amino acids, therefore, indicates that DOM is more refractory. Similarly, the ratio of glutamic acid to GABA (Glu/GABA) is one of the biodegradation indices since GABA, a non-protein amino acid, is accumulated by the degradation of glutamic acid during microbial degradation 30 . As mentioned above, the averaged D/L ratios of amino acids increased gradually according to the red tide stages in the study region (one-way ANOVA: p = 0.008) (Fig. 5). The ratios of Glu/GABA were also higher in the early stage of red tide (average: 13 ± 4) than those for the other periods (one-way ANOVA: p = 0.021) (Fig. 5). Thus, it is likely that fresh amino acids decrease in the course of the red tide succession.
THAA (%DON) has been widely used for characterizing the bioavailability of DOM [70][71][72] . Our results show that the yields of THAA (%DON) in the early stage of red tide (average: 8 ± 3%) were significantly higher than those for the other periods (Fig. 5). This trend consistently indicates that bioavailable DON concentrations were highest in the early stage of red tide and diminished in the final stage (Fig. 5). Therefore, all biogeochemical parameters (D/L ratio, Glu/GABA, and THAA [%DON]) suggest that the amount of bioavailable DOM is critical for the occurrence of red tides if other conditions (i.e., depleted inorganic nutrients, temperature, and salinity) are favorable for red tide outbreaks. ). Our results reveal that freshly produced DOM plays an important role in the red tide outbreak, together with other environmental conditions. Therefore, our tools for determining the sources and origins of DOM in this red tide region can be utilized similarly in other red tide areas of the global ocean. www.nature.com/scientificreports/

Data availability
The datasets analysed during the current study are available from the corresponding author on reasonable request. www.nature.com/scientificreports/